The endogenous hematopoetic growth factor, granulocyte-colony stimulating factor (G-CSF, synonym “colony-stimulating factor 3”=CSF3) regulates the proliferation and differentiation of progenitor cells within the bone marrow and the release of mature neutrophilic granulocytes (“neutrophils”) into the peripheral blood. Cancer chemotherapy, which affects rapidly dividing cells, frequently leads to a side effect termed “neutropenia”. Neutropenia is a decrease in counts of neutrophils in the peripheral blood and affects more than one of three patients receiving myelosuppressive chemotherapy for cancer. Patients driven into neutropenia can develop fever (“febrile neutropenia”) and have an increased risk for infections. Life-threatening gastrointestinal and pulmonary infections occur, as does sepsis. A subsequent cycle of chemotherapy may have to be delayed until the patient has recovered from neutropenia. Recombinant human G-CSF is an effective pharmaceutical substance and successfully applied to treat chemotherapy-induced neutropenia. It restores the number of neutrophils in the blood and keeps it above the critical level (Dale 2002).
Natural human G-CSF is an O-glycosylated protein consisting of 174 amino acids and is relatively hydrophobic. The carbohydrate chain in the glycosylated form is located at threonine 133. Besides this major form another splice form can occur in vivo which bears additional three amino acids (Zsebo 1986). When recombinant human G-CSF is expressed in E. coli, the following can be observed: First, the recombinant protein is produced in inclusion bodies; second, the resulting G-CSF molecule is devoid of the natural carbohydrate chain, and third, the recombinant G-CSF bears an additional, N-terminal methionine. This G-CSF molecule, designated N-methionyl-G-CSF or rmet(hu)G-CSF, received the international non-proprietary name (INN) “filgrastim” and has a molecular weight of 18.7-18.9 kD (Welte 1996). The theoretical relative mass of filgrastim (Mr) is 18.799. The G-CSF polypeptide chain contains five cysteines and structural investigations with filgrastim revealed two disulfide bonds between Cys 37-43 and Cys 65-75, while the unpaired Cys 18 remains reduced (Wingfield 1988). The first product on the market was Amgen's Neupogen® containing filgrastim, an E. coli-expressed, recombinant human met-G-CSF (Welte 1996). Another G-CSF product, approved in the European Union, Chugai's Granceyte®, containing lenograstim, is derived from recombinant mammalian (CHO) cells and is glycosylated (Holloway 1996). In addition, Amgen launched in 2002 an improved version of G-CSF, Neulasta®, which consists of a conjugate filgrastim and polyethylene glycol (INN pegfilgrastim) (Molineux 2004). Finally, several biosimilar versions of Neupogen® were launched in Europe by different generic pharmaceutical companies during the last years.
Overexpression of heterologous recombinant polypeptides in transformed microorganisms often results in the formation of so-called inclusion bodies (IBs), which contain the recombinant protein. These inclusion bodies are highly refractile, amorphous aggregates and the polypeptides therein are generally unfolded, reduced, inactive, and at least partially insoluble in common aqueous buffers. Processes for obtaining recombinant proteins from inclusion bodies are described in the art and generally comprise lysis and disruption of the cells followed by centrifugation. The pellet comprising a large proportion of inclusion bodies is usually washed with detergents to remove lipid membranes, lipopolysaccharides (LPS) and other cell debris or contaminants.
The scientific literature provides many methods how such inclusion bodies can be isolated from bacteria and purified and how the recombinant protein afterwards can be solubilised and refolded into its native state. (The terms ‘refolding’ and ‘renaturation’ are synonymously used herein).
Different strategies have been used to solubilise the recombinant protein. Besides ionic or non-ionic detergents, such as sodium dodecyl sulfate (SDS) or N-laurylsarcosin (sarkosyl), chaotropic reagents, such as guanidine hydrochloride (GuHCl) or urea, have been used to solubilise a protein of interest. Often the solubilisation is performed under alkaline conditions (pH 8-12.5) in presence of reducing agents, such as dithiothreitol (DTT), dithicerythrol (DTE) or 2-mercaptoethanol (ME) (Marston 1986, Rudolph 1990, Rudolph 1996, Dietrich 2003). Typically, the solubilised protein is at first fully reduced and inactive; and then undergoes refolding prior to the chromatographic purification.
For example, EP0219874 discloses generic methods for refolding of recombinant proteins from E. coli inclusion bodies. For the solubilisation the chaotropic agents GuHCl and arginine were used at high pH. EP0219874 describes the formation of disulfide bridges under redox conditions provided by GSH/GSSG.
Rudolph 1990 describes the following sequence of steps: a) the use of GuHCl or urea for solubilisation at pH 8-9 under reductive conditions (DTT, DTE or 2-ME), b) removal of reagents by dialysis or gel chromatography (Sephadex G-25) and c) disulfide formation (=refolding) by oxido shuffling systems or by reversal chemical modification of protein thiols, both based on the effect of added GSH/GSSG.
Another review (Rudolph 1996) put emphasis on additives used during refolding which can affect the solubility and stability of the unfolded protein, the folding intermediates and the native folded protein. The authors suggest a generic basic protocol for solubilisation and refolding: Solubilisation with 6M GuHCl and 100 mM DTT at pH 8. Reducing agents are removed by dialysis and pH is adjusted to 4.5. Folding is performed by high dilution (1:200) in a buffer with EDTA and GSH/GSSG, at pH 7.5 to 8.5.
Dietrich 2003 describes the solubilisation of proteins from E. coli inclusion bodies with 6M GuHCl under reductive conditions (DTE). The refolding incubation was defined at pH 9 in 1 M arginine in presence of GSH/GSSG. Final purification was performed using hydrophobic interaction chromatography (HIC) followed by cation exchange chromatography (CEX) using SP Sepharose.
An application note available from GE Healthcare 2007 (Application Note 18-1112-33, 1-4) also reviewed general protocols. Solubilisation is recommended with 8M urea or 6M GuHCl. Refolding was mentioned as slow dialysis or dilution near neutral pH. Alternatively a chromatographic step can be used for refolding. The suggested chromatographic methods comprise size exclusion chromatography (SEC), ion exchange chromatography (IEX) and hydrophobic interaction chromatography (HIC) which is suggested instead of dialysis or dilution.
WO00/02901 describes a general method for refolding by applying high pressure within a refolding tank. Optionally, chaotropic agents and/or redox compounds (DTT/GSSG) are present in the refolding buffers.
Starting in the 1980s there is a long history of developing methods for producing biologically active recombinant G-CSF. The majority of publications describe production in E. coli. In this host, G-CSF is well-expressed and accumulates normally in inclusion bodies. Other expression systems used were for example CHO cells (Holloway 1994), human cells (WO01/04154), or yeast (U.S. Pat. No. 5,055,555).
Zsebo 1986 described the solubilisation of G-CSF with 2% sarkosyl and the purification of soluble G-CSF by AEX and CEX chromatographies.
WO87/01132 describes E. coli-derived human G-CSF (filgrastim). Two alternative methods for refolding/purification were described: Process 1): G-CSF was solubilised with 1% lauric acid (a saturated C15 fatty acid) and oxidized with 40 μM CuSO4 followed by HPLC-purification on a C4 material. Process 2): Solubilisation was performed with 2% sarkosyl and oxidation with 20 μM CuSO4. The G-CSF was precipitated with acetone, again solubilised with 6M GuHCl and by this condition unfolded. After removal of GuHCl by gel chromatography (Sephadex G-25, a refolding step) the subsequent chromatography was CEX (CM-Cellulose) followed by a final size exclusion chromatography (SEC, Sephadex G-75).
An alternative solubilising agent was used by Devlin 1988, which solubilised the IB-pellet with 10% SDS and purified the SDS-loaded G-CSF by SEC (Sephacryl S-200 in 0.1% SDS) followed by reversed-phase high pressure liquid chromatography (RP-HPLC, Vydac C4).
These early publications focused on constructing suitable expression systems and getting purified substance for further characterizing of G-CSF, rather than providing refolding and purification processes suitable for commercial large scale production of recombinant G-CSF. A more advanced process was published in WO89/10932, which describes methods for purification of human and bovine G-CSF from E. coli IBs. The IBs were treated with detergents (deoxycholate) to extract contaminants. Sarkosyl was used to solubilise G-CSF. Oxidation was performed, with CuSO4. Further processes were described in Lu 1992 and Heidari 2001.
Several alternatives to the above mentioned methods for solublisation and oxidative refolding, including the method of WO89/10932 using 2% sarkosyl/40 μM CuSO4, have been published. Most of these strategies followed the classical general approach of solubilisation (Marston 1986, Rudolph 1990, Rudolph 1996, see above), using strong denaturants such as GuHCl and urea, completely breaking the hydrogen bonds under reductive conditions at alkaline pH. Especially the more recent publications preferred the refolding of GuHCl— or urea-solubilised G-CSF.
For example, Wingfield 1988 describes the purification of a wild-type G-CSF and a mutein from E. coli IBs. Solubilisation was performed with 6M GuHCl. A first purification was done with unfolded protein on SEC (Sephacryl S200) in presence of 4M GuHCl. G-CSF was then oxidized and refolded by dialysis against 3 M urea and further purified with CEX (CM-Sepharose) and SEC (Ultrogel AcA54).
A paper from Kang 1998 described N-meth-hu-G-CSF expression in E. coli IBs. G-CSF was solubilised with 2M urea under alkaline conditions. Refolding was initiated by dilution and incubation for 16 h at room temperature. Then the pH was lowered to pH 5.5 and the emerging precipitates were removed. Two subsequent chromatographies were performed, a CEX step (SP Sepharose) was followed by a chromatofocusing step (PBE94) using polybuffers.
WO98/53072 discloses a G-CSF bearing a small leader peptide at the N-terminus which was expressed in E. coli without cleavage of the signal peptide and thus accumulated in IBs. Solubilisation was performed in 8M urea under reductive conditions (10 mM DTT). The solubilised. G-CSF was subjected to AEX (DEAE-Sepharose) followed by SEC (Sephacryl 200). An oxidative refolding was described which comprises a rather short incubation in presence of 2 mM GSH.
In another publication (Wang 2005) the inclusion bodies were solubilised with 8M urea in presence of 100 mM 2-ME, and 5 mM EDTA at pH 8. A matrix-bound refolding was performed during the subsequent AEX chromatography (Q-Sepharose). G-CSF was bound to the column and while staying bound the urea concentration in the mobile phase was lowered. The buffer contained GSH/GSSG and the eluted G-CSF was biologically active. Further methods are described in WO01/87925 and WO02004/015124.
WO2004/001056 discloses a method comprising a first incubation at pH 8 for 6 hours followed by a second incubation at pH 4-5 for 6-8 hours.
WO2006/097944 describes that IBs are solubilised with urea or GuHCl (2-6M) at alkaline pH (8-11) and the refolding was performed after dilution at acidic ph for 6-16 h at room temperature.
WO2006/135176 deals with G-CSF muteins which were purified for subsequent PEGylation. The G-CSF variants were expressed in E. coli and solubilised from IBs by using 8M urea at pH 11. Refolding was performed by diluting to 2M urea and 50 mM glycine and incubated at pH 9 over night.
EP1630173 discloses methods for isolating and refolding of G-CSF (filgrastim) from E. coli IBs. The methods are based on extraction with denaturants, preferentially GuHCl. Refolding was performed in the presence of GSH/GSSG, high pH and low temperatures.
EP1837346 describes methods for isolating, refolding and purifying G-CSF (filgrastim) expressed in E. coli IBs. GuHCl was used for solubilisation and the refolding was performed in presence of GSH. A subsequent gel chromatography (Sephadex G-25) was applied for removal of denaturants and buffer.
Rao 2008 describes a process for the production of G-CSF from E. coli IBs. The IBs were dissolved in 2M urea in presence of 25 mM cysteine at unusually high pH values (pH 12-12.8).
Similar methods for solubilisation, refolding and purification of G-CSF (filgrastim) were described by Vanz 2008.
Khalilzadeh 2008 suggested modifications to the method using sarkosyl/CuSO4 described above. The solubilisation of washed IBs was performed with 8M urea. Refolding was performed by step dialysis to reduce the urea from 8M to 0M. CuSO4 concentration ranged from 5-60 μM, and an optimum was shown for 40 μM. The chromatographic sequence consists of three steps, AEX (DEAE) followed by HIC (Butyl) and a final SEC (Sephadex G-25).
WO2008/096370 also describes refolding and purification of huG-CSF from E. coli IBs. G-CSF was solubilised with urea in presence of DTT and pH was raised to pH 12-12.5.
Finally, WO2010/146599 discloses solubilisation of G-CSF with 6M GuHCl and reduction with DTT. For refolding a complex buffer was composed consisting of 2M urea, 0.1M arginine, 10% sucrose, 2 mM EDTA and including oxido-shuffling agents such as 10 mM Na-ascorbate/dihydroascorbate/DTT or GSH/GSSG or cysteine/cystine (redox).
There is an ongoing need for new methods for obtaining G-CSF from inclusion bodies.